Injury-elicited differential transcriptional regulation of phospholipid growth factor receptors in the cornea (original) (raw)
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Phospholipid Growth Factors and Corneal Wound Healing
Annals of the New York Academy of Sciences, 2006
In many tissue types, wound healing involves cell division and migration over and into the wound area to cover and remodel the wound. LPA and other members of the phospholipid lipid growth factor (PLGF) family stimulate many of the activities involved in wound healing. In the rabbit cornea, we have found that keratocytes from wounded corneas have a volumeactivated Cl − current activated by LPA and alkenyl-LPA. This current is minimally activated by cyclic PA and SPC, and is not activated by LPA in cells from uninjured corneas. Biochemical examination of PLGFs in aqueous humor and lacrimal fluid before and after wounding identified LPA, alkenyl-GP, PA, and lyso PS, with elevated PLGF activity after wounding. In recent experiments examining human corneal cell lines and cultured cells using RT-PCR, we found mRNA for EDG receptors 1-5, with an apparent increase in EDG-3,-4, and-5 following brief SDS application to cell lines, and EDG receptors 2-5 induction in late-passage human corneal epithelial cells. This work points to a significant role for PLGFs in the corneal wound-healing process.
Cellular Signalling, 2012
Granular corneal dystrophy type 2 (GCD2) is an autosomal dominant disease caused by a R124H point mutation in the transforming growth factor-β-induced gene (TGFBI). However, the cellular role of TGFBI and the regulatory mechanisms underlying corneal dystrophy pathogenesis are still poorly understood. Lysophosphatidic acid (LPA) refers to a small bioactive phospholipid mediator produced in various cell types, and binds G protein-coupled receptors to enhance numerous biological responses, including cell growth, inflammation, and differentiation. LPA levels are elevated in injured cornea and LPA is involved in proliferation and wound healing of cornea epithelial cells. Accumulating evidence has indicated a crucial role for LPA-induced expression of TGFBI protein (TGFBIp) through secretion of transforming growth factor-beta1 (TGF-β1). In the current study, we demonstrate that LPA induces TGFBIp expression in corneal fibroblasts derived from normal or GCD2 patients. LPA-induced TGFBIp expression was completely inhibited upon pretreatment with the LPA 1/3 receptor antagonists, VPC32183 and Ki16425, as well as by silencing LPA 1 receptor expression with small hairpin RNA (shRNA) in corneal fibroblasts. LPA induced secretion of TGF-β1 in corneal fibroblasts, and pretreatment with the TGF-β type I receptor kinase inhibitor SB431542 or an anti-TGF-β1 neutralizing antibody also inhibited LPA-induced TGFBIp expression. Furthermore, we show that LPA requires Smad2/3 proteins for the induction of TGFBIp expression. LPA elicited phosphorylation of Smad2/3, and Smad3 specific inhibitor SIS3 or siRNA-mediated depletion of endogenous Smad2/3 abrogates LPA-induced TGFBIp expression. Finally, we demonstrate that LPA-mediated TGFBIp induction requires JNK activation, but not ERK signaling pathways. These results suggest that LPA stimulates TGFBIp expression through JNK-dependent activation of autocrine TGF-β1 signaling pathways and provide important information for understanding the role of phospholipids involved in cornea related diseases.
Expression of Phospholipases A2 and C in Human Corneal Epithelial Cells
2004
To achieve a better understanding of the involvement of phospholipases in the inflammation and wound-healing processes in human corneal epithelial cells (HCECs), expression of phospholipase A 2 s (PLA 2 s) and phospholipase Cs (PLCs) was examined in the human corneal epithelium. METHODS. Specific primers were designed for RT-PCR amplification of the known secreted (s)PLA 2 , cytosolic (c)PLA 2 , and PLC mRNAs. Corresponding PCR products were cloned and the DNA sequenced. Immunofluorescence of flatmounted corneal sections and Western blot analyses were used to detect the PLA 2 s and PLCs expressed by HCECs. RESULTS. The mRNAs for the following phospholipases were detected by RT-PCR in the HCECs: sPLA 2 GIII, -GX, and -GXIIA; cPLA 2 ␣ and -␥; PLC1
Lysophospholipid Receptor-Mediated Calcium Signaling in Human Keratinocytes
Journal of Investigative Dermatology, 2008
The lysophospholipids, sphingosine-1-phosphate (S1P) and lysophosphatidic acid (LPA), stimulate chemotaxis and induce differentiation of human keratinocytes. As Ca 2 þ plays an important role in keratinocyte differentiation, we studied Ca 2 þ signaling by S1P and LPA in these cells, known to express mRNA transcripts of the S1P 1À5 and LPA 1À3 receptors, and the receptor subtypes involved in this process. S1P and LPA caused transient increases in intracellular free Ca 2 þ concentration ([Ca 2 þ ] i ), with pEC 50 values of 8.5 ± 0.11 and 7.5 ± 0.23, respectively. The [Ca 2 þ ] i increases are apparently mediated by stimulation of phospholipase C and involve Ca 2 þ mobilization from thapsigargin-sensitive stores and subsequent Ca 2 þ influx. The LPA-induced [Ca 2 þ ] i increases were not inhibited by the LPA 1/3 receptor antagonist, dioctanoylglycerol pyrophosphate. The S1P-induced [Ca 2 þ ] i increases were largely inhibited by the putative S1P 3 antagonist, BML-241, and the S1P 1/3 antagonist, VPC23019. The S1P 1 -specific agonist, SEW2871, did not increase [Ca 2 þ ] i but stimulated chemotaxis of keratinocytes, which was fully blocked by S1P 1 antisense oligonucleotides. The data indicate that LPA and S1P potently increase [Ca 2 þ ] i in human keratinocytes and that the effect of LPA is mediated by LPA 2 , whereas that of S1P is mediated at least to a large part by S1P 3 . The S1P 1 receptor, without stimulating [Ca 2 þ ] i increases, mediates chemotaxis of keratinocytes.
Archives of Dermatological Research, 2004
Human keratinocytes are exposed to strong physical changes, and have the potentiality to react to external stimuli by switching on adaptation mechanisms. In hyperosmotically shocked keratinocytes a rapid and strong increase in calcium has been observed. We showed that this increase could not be prevented by growing the cells in medium devoid of calcium and in the presence of EGTA, indicating that the intracellular calcium increase was due to delivery from internal stores. Further, we observed an increased synthesis of dyacylglycerol and inositol trisphosphates after shock, suggesting that phospholipase C mediates both events. Our experiments demonstrated that osmotic shock in human keratinocytes leads to activation of phospholipase C-γ1, as measured using an in vitro assay system. This activation is independent of protein tyrosine phosphorylation and corresponded to a relocation of the enzyme to perinuclear membranes as shown by immunofluorescence.
Current Eye Research, 1998
Purpose. Activation of phospholipase D (PLD) is believed to be an important signaling pathway involved in cell growth and differentiation in several tissues, in response to a variety of mitogens. The aim of the present study was to investigate the effect of epidermal growth factor (EGF) on PLD activity in rabbit corneal epithelial cells (RCEC). We have also examined whether the EGF effect is dependent on concurrent activation of phospholipase C (PLC), protein kinase C (PKC) or phosphatidylinositol 3-kinase (PI 3-kinase) in these cells. Methods. RCEC, immortalized with adenovirus SV-40, were cultured until they became confluent. The cells were labeled with [ 3 H]myristic acid and incubated with or without EGF or other agents for specified time intervals. PLD activity was measured by quantifying [ 3 H]phosphatidylethanol in cells incubated in the presence of ethanol. PLC activity was determined by measuring the radioactivity in inositol trisphosphate in myo[ 3 H]inositol-labeled RCEC. PI 3-kinase activity was assessed by measuring the production of PIP 3 in 32 P-labeled cells. Results. Addition of EGF to RCEC stimulated PLD activity in a time-and dose-dependent manner. The maximal effect was observed with 150 ng/ml EGF and at 10 min of incubation. The PLD activity was also stimulated when phorbol myristate acetate (PMA) was added to the cells. Treatment of the cells with EGF stimulated PLC activity which was inhibited by U73122, a PLC inhibitor. Under the same experimental conditions, the inhibitor had no effect on EGF-stimulated PLD activity. Downregulation of PKC or treatment of the cells with RO31-8220, a PKC inhibitor, inhibited the PMA-but not EGF-stimulated PLD activity. Incubation of the cells with wortmannin, a PI 3-kinase inhibitor, abolished the EGF-stimulated PI 3-kinase activity, but potentiated the EGF-stimulated PLD activity. The EGF effect was inhibited by treatment of the cells with tyrphostin B 42 , a receptor tyrosine kinase inhibitor. Conclusions. These results indicate that EGF stimulates PLD activity in RCEC by a mechanism that involves tyrosine phosphorylation of a protein(s) in the cascade of biochemical reactions initiated by EGF-receptor interaction, and it is not dependent on concurrent activation of PKC, PLC, or PI 3-kinase in these cells. Curr. Eye Res. 17: 294-300, 1998.
Cell wounding activates phospholipase D in primary mouse keratinocytes
The Journal of Lipid Research, 2013
characterized by growth arrest and expression of the mature keratins 1 and 10 in the fi rst differentiated layer of the epidermis, the spinous layer. Early differentiation in the spinous layer is followed by further differentiation in the granular layer, which is accompanied by expression of proteins that are essential for the formation of the cornifi ed envelope and corneocytes. The corneocytes constitute the outer layer of the epidermis, the stratum corneum, and give skin its resilience to mechanical stress (as reviewed in Ref. 1). Defi ciencies in the mechanical barrier function of the epidermis result in skin diseases. For example, epidermolysis bullosa simplex and epidermolytic hyperkeratosis arise through mutations in keratins comprising the intermediate fi laments and are characterized by extensive blistering and epidermal sloughing as a result of the mechanical stresses encountered by routine interactions with the environment (as reviewed in Ref. 2). Many tissues of the body in addition to the skin are exposed to mechanical stresses that result in tearing, or disrupting, the plasma membrane of the constituent cells. These disruptions will result in cell death if left unrepaired. However, cells possess an active plasma membrane repair process that can restore plasma membrane integrity if the disruption is not too extensive (as reviewed in Ref. 3). For example, intestinal cells in the gastrointestinal tract are subjected to mechanical perturbations during the transit of a food bolus; these plasma membrane disruptions can be repaired to allow cell survival (4-6). Similarly, eccentric contraction of skeletal muscle as a result of downhill treadmill running induces plasma membrane disruptions that are largely repaired (7). Routine ambulation also appears to lead to plasma membrane disruptions in the epidermis of the digits (8). Therefore, it is critical that cells in these mechanically active tissues be able to repair membrane Abstract Plasma membrane disruptions occur in mechanically active tissues such as the epidermis and can lead to cell death if the damage remains unrepaired. Repair occurs through fusion of vesicle patches to the damaged membrane region. The enzyme phospholipase D (PLD) is involved in membrane traffi ckiing; therefore, the role of PLD in membrane repair was investigated. Generation of membrane disruptions by lifting epidermal keratinocytes from the substratum induced PLD activation, whereas removal of cells from the substratum via trypsinization had no effect. Pretreatment with 1,25-dihydroxyvitamin D 3 , previously shown to increase PLD1 expression and activity, had no effect on, and a PLD2-selective (but not a PLD1-selective) inhibitor decreased, cell lifting-induced PLD activation, suggesting PLD2 as the isoform activated. PLD2 interacts functionally with the glycerol channel aquaporin-3 (AQP3) to produce phosphatidylglycerol (PG); however, wounding resulted in decreased PG production, suggesting a potential PG defi ciency in wounded cells. Cell lifting-induced PLD activation was transient, consistent with a possible role in membrane repair, and PLD inhibitors inhibited membrane resealing upon laser injury. In an in vivo full-thickness mouse skin wound model, PG accelerated wound healing. These results suggest that PLD and the PLD2/AQP3 signaling module may be involved in membrane repair and wound healing.-Arun, S.
Molecular vision, 2013
To quantitatively assess the superoxide dismutase 1 (SOD1), transforming growth factor, beta 1 (TGF-β1), and dual-specificity phosphatase 1 (DUSP1) messenger ribonucleic acid (mRNA) expression levels as the main intracellular reactive oxygen species neutralizers, wound healing mediators, and immunomodulators (respectively) in keratoconic (KCN) and non-KCN corneas. Total RNA was extracted from normal and keratoconic cultured corneal stromal fibroblasts. Semiquantitative reverse transcriptase polymerase chain reaction (RT-PCR) was used to measure the relative expression levels of mRNAs of the SOD1, TGF-β1, and DUSP1 genes. The mRNA expression of TGF-β1 and DUSP1 was augmented in the KCN corneas (three- and fivefold, respectively; both p<0.05). The KCN and non-KCN samples showed no difference in comparative SOD1 mRNA levels. This study demonstrated a higher level of DUSP1 and TGF-β1 expression as known molecules in the inflammatory process. These results may provide new insight into...